Quantifying cathepsin S activity in antigen presenting cells using a novel specific substrate.
Cathepsin S (CatS) is a lysosomal cysteine protease belonging to the papain superfamily. Because of the relatively broad substrate specificity of this family, a specific substrate for CatS is not yet known. Based on a detailed study of the CatS endopeptidase specificity, using six series of internally quenched fluorescent peptides, we were able to design a specific substrate for CatS. The peptide series was based on the sequence GRWHTVGLRWE-Lys(Dnp)-DArg-NH2, which shows only one single cleavage site between Gly and Leu and where every substrate position between P-3 and P-3' was substituted with up to 15 different amino acids. The endopeptidase specificity of CatS was mainly determined by the P-2, P-1', and the P-3' substrate positions. Based on this result, systematically modified substrates were synthesized. Two of these modified substrates, Mca-GRWPPMGLPWE-Lys(Dnp)-DArg-NH2 and Mca-GRWHPMGAPWE-Lys(Dnp)-DArg-NH2, did not react with the purified cysteine proteases cathepsin B (CatB) and cathepsin L (CatL). Using a specific CatS inhibitor, we could further show that these two peptides were not cleaved by endosomal fractions of antigen presenting cells (APCs), when CatS was inhibited and related cysteine proteases cathepsin B, H, L and X were still active. Although aspartic proteases like cathepsin E and cathepsin D were also present, our substrates were suitable to quantify cathepsin S activity specifically in APCs, including B cells, macrophages, and dendritic cells without the use of any protease inhibitor. We find that CatS activity differs significantly not only between the three types of professional APCs but also between endosomal and lysosomal compartments.
Lützner
Nicolas
N
Interfaculty Institute of Biochemistry, Medical and Natural Sciences Research Centre, University of Tuebingen, Tuebingen, Germany.
Kalbacher
Hubert
H
D000595:Amino Acid Sequence; D000938:Antigen-Presenting Cells / Q000378:metabolism*; D002402:Cathepsin D / Q000032:analysis; D020059:Cathepsin E / Q000032:analysis; D002403:Cathepsins / Q000032:analysis* / Q000737:chemistry*; D010450:Endopeptidases / Q000737:chemistry; D011992:Endosomes / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008247:Lysosomes / Q000737:chemistry; D008264:Macrophages / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity
D010455:Peptides; D011994:Recombinant Proteins; D002403:Cathepsins; D010450:Endopeptidases; C028217:cathepsin S; D020059:Cathepsin E; D002402:Cathepsin D